While gels can be used to eliminate adapter dimers and contaminating RNAs, there is still a possibility for high prevalence in sequencing reads even after gel excision. On the above left, QIAseq miRNA shows a robust miRNA library with no adapter dimers or contaminating RNA after the basic protocol that includes a bead-based purification. Compared to libraries generated with competitor kits (prior to a required tedious gel excision), the QIAseq-derived miRNA library is much more robust and devoid of adapter dimers and contaminating RNAs.

Starting with total RNA isolated from any sample, the entire QIAseq miRNA Library Kit workflow can be completed in 7 hours. Molecular barcodes are attached during the reverse-transcription reaction. Thus, any library amplification and sequencing biases can be accounted for.

Robust detection of miRNA

The QIAseq miRNA Library Kit has been designed to enhance yields from biofluids such as serum. This figure shows robust detection of miRNA from serum samples.

A huge issue with miRNA sequencing workflows is the presence of adapter dimer contamination. The QIAseq miRNA Library Kit has fully optimized library process to virtually eliminate adapter dimerization, even from very low inputs of total RNA (see figure Adapter dimers (AD) and contaminating RNA steal your reads during miRNAseq experiments). In addition, the reaction chemistry facilitates the preparation of robust, miRNA-focused libraries while all but eliminating biases and background contaminants. Together, these benefits highlight that the QIAseq miRNA Library Kit is designed to maximize the yield of miRNA available to sequence.

Due to the growth of circulating miRNAs as potential biomarkers, the QIAseq miRNA Library Kit is optimized to map miRNA down to ultralow input levels. In addition, the kit integrates Unique Molecular Indices (UMIs) into the reverse-transcription process, enabling unbiased and accurate miRNome-wide quantification of mature miRNAs by NGS. Collectively, QIAseq miRNA offers an unrivaled Sample to Insight solution for differential expression analysis and discovery of novel miRNAs using next-generation sequencing (see figure QIAseq miRNA workflow).

Quantification of miRNA expression can be performed using a variety of technologies including next-generation sequencing (NGS) and real-time PCR (qPCR). While NGS is the default tool for novel miRNA discovery, commercially available library preparation kits are tedious and introduce biases. As a result, qPCR has been the “go to” technology for quantification of miRNA expression, until now. QIAseq miRNA defines a new generation in small RNA sequencing products and includes several distinct features not found in other sequencing kits. With the QIAseq miRNA Library Kit, the power of NGS has been combined with single molecule quantification from UMIs to produce the most representative expression data possible.

Procedure

QIAGEN offers a true Sample to Insight workflow, from sample isolation to data analysis and interpretation. Total RNA is first extracted from biofluids (such as serum, plasma, CSF and urine), cells, fresh/frozen tissues or FFPE tissues using a miRNeasy kit. From there, miRNA sequencing libraries are prepared using the QIAseq miRNA Library Kit (see figure Under a day prep).

In an unbiased reaction, adapters are ligated sequentially to the 3’ and 5’ ends of miRNAs. Subsequently, universal cDNA synthesis with UMI assignment, cDNA cleanup, library amplification and library cleanup are performed. Proprietary methodology, using modified oligonucleotides, virtually eliminates the presence of adapter dimers in the sequencing library and effectively removes a major contaminant often observed during sequencing. Bead-based cleanups eliminate the majority of unwanted background noise that steals sequencing reads from a budget. The UMIs ensure that during data analysis, the sample is analyzed specifically, not amplification or sequencing artifacts. To go from sample to sequencer, the process takes only eight hours, with minimal hands-on time. Up to a maximum of 48 samples can be multiplexed.
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After sequencing, “.fastq” or “.fastq.gz” file formats can be uploaded directly to the GeneGlobe Data Analysis Center for primary mapping and molecular tag counting. For well-characterized species, such as human, mouse and rat, reads are mapped to species-specific miRBase and genome databases. For poorly-characterized or novel species, read are mapped to the entire miRBase database. Secondary differential expression analysis is then performed with multiple methods for molecular tag count normalization and visualization of the resulting data.

Applications

Biofluids (such as serum, plasma, CSF and urine), cells, fresh/frozen tissue and FFPE tissues containing total and miRNA are compatible with the QIAseq miRNA Library Kit. Use the QIAseq miRNA to characterize the next circulating miRNA biomarker. Uncover miRNA signatures locked away in FFPE tissues. Completely characterize a new species. The QIAseq miRNA Library Kit has been designed to enhance yields from biofluids such as serum. In the figure Detection of miRNA, the QIAseq miRNA Library Kit shows robust detection of miRNA from serum samples. Mapped reads were then compared to adapter dimers in serum samples. QIAseq miRNA still shows superior mapping of miRNAs even with limited samples (see figure Read distribution in serum samples). With the QIAseq miRNA Library Kit, determine the differential expression of any known or novel miRNAs from any total RNA sample derived from any species.

QIAseq miRNA is the ultimate tool to enhance discovery and expression from large-scale projects with hundreds of samples down to the small pilot focused on a group of target miRNA. QIAseq miRNA offers an unrivaled Sample to Insight solution for differential expression analysis and discovery of novel miRNAs using next-generation sequencing.